EPO primary response gene, EPRG3pt

ABSTRACT

EPRG3pt polypeptides and polynucleotides and methods for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods for utilizing EPRG3pt polypeptides and polynucleotides in therapy, and diagnostic assays for such.

[0001] This application claims the benefit of earlier filed U.S.Provisional Application No. 60/054,464, filed Aug. 1, 1997, whosecontents are herein incorporated by reference in their entirety.

FIELD OF THE INVENTION

[0002] This invention relates to newly identified polypeptides andpolynucleotides encoding such polypeptides, to their use in therapy andin identifying compounds which may be agonists, antagonists and/orinhibitors which are potentially useful in therapy, and to production ofsuch polypeptides and polynucleotides.

BACKGROUND OF THE INVENTION

[0003] The drug discovery process is currently undergoing a fundamentalrevolution as it embraces functional genomics′, that is, high throughputgenome- or gene-based biology. This approach is rapidly supersedingearlier approaches based on ′positional cloning′. A phenotype, that is abiological function or genetic disease, would be identified and thiswould then be tracked back to the responsible gene, based on its geneticmap position.

[0004] Functional genomics relies heavily on the various tools ofbioinformatics to identify gene sequences of potential interest from themany molecular biology databases now available. There is a continuingneed to identify and characterise further genes and their relatedpolypeptides/proteins, as targets for drug discovery.

SUMMARY OF THE INVENTION

[0005] The present invention relates to EPRG3pt, in particular EPRG3ptpolypeptides and EPRG3pt polynucleotides, recombinant materials andmethods for their production. In another aspect, the invention relatesto methods for using such polypeptides and polynucleotides, includingthe treatment of anemia, polycythemia, cancer, AIDS, and drug-inducedanemias, hereinafter referred to as “the Diseases”, amongst others. In afurther aspect, the invention relates to methods for identifyingagonists and antagonists/inhibitors using the materials provided by theinvention, and treating conditions associated with EPRG3pt imbalancewith the identified compounds. In a still further aspect, the inventionrelates to diagnostic assays for detecting diseases associated withinappropriate EPRG3pt activity or levels.

DESCRIPTION OF THE INVENTION

[0006] In a first aspect, the present invention relates to EPRG3ptpolypeptides. Such peptides include isolated polypeptides comprising anamino acid sequence which has at least 70% identity, preferably at least80% identity, more preferably at least 90% identity, yet more preferablyat least 95% identity, most preferably at least 97-99% identity, to thatof SEQ ID NO:2 over the entire length of SEQ ID NO:2. Such polypeptidesinclude those comprising the amino acid of SEQ ID NO:2.

[0007] Further peptides of the present invention include isolatedpolypeptides in which the amino acid sequence has at least 70% identity,preferably at least 80% identity, more preferably at least 90% identity,yet more preferably at least 95% identity, most preferably at least97-99% identity, to the amino acid sequence of SEQ ID NO:2 over theentire length of SEQ ID NO:2. Such polypeptides include the polypeptideof SEQ ID NO:2.

[0008] Further peptides of the present invention include isolatedpolypeptides encoded by a polynucleotide comprising the sequencecontained in SEQ ID NO:1.

[0009] Polypeptides of the present invention are believed to bestructurally related to other proteins having homology and/or structuralsimilarity with human p27 (Rasmussen, U.B., et al., 1993, CancerResearch 53:4096-4101). They are therefore of interest because theexpression of certain proteins is induced by EPO, and the presence ofEPO is required for maintenance of expression of such proteins. TheseEPO-related proteins are involved in the proliferation of EPO-dependentcells and may be important in the growth and development of erythroidand other hematopoietic lineages. These properties are hereinafterreferred to as “EPRG3pt activity” or “EPRG3pt polypeptide activity” or“biological activity of EPRG3pt”. Also included amongst these activitiesare antigenic and immunogenic activities of said EPRG3pt polypeptides,in particular the antigenic and immunogenic activities of thepolypeptide of SEQ ID NO:2. Preferably, a polypeptide of the presentinvention exhibits at least one biological activity of EPRG3pt.

[0010] The polypeptides of the present invention may be in the form ofthe “mature” protein or may be a part of a larger protein such as afusion protein. It is often advantageous to include an additional aminoacid sequence which contains secretory or leader sequences,pro-sequences, sequences which aid in purification such as multiplehistidine residues, or an additional sequence for stability duringrecombinant production.

[0011] The present invention also includes include variants of theaforementioned polypeptides, that is polypeptides that vary from thereferents by conservative amno acid substitutions, whereby a residue issubstituted by another with like characteristics. Typical suchsubstitutions are among Ala, Val, Leu and Ile; among Ser and Thr; amongthe acidic residues Asp and Glu; among Asn and Gln; and among the basicresidues Lys and Arg; or aromatic residues Phe and Tyr. Particularlypreferred are variants in which several, 5-10, 1-5, 1-3, 1-2 or 1 aminoacids are substituted, deleted, or added in any combination.

[0012] Polypeptides of the present invention can be prepared in anysuitable manner. Such polypeptides include isolated naturally occurringpolypeptides, recombinantly produced polypeptides, syntheticallyproduced polypeptides, or polypeptides produced by a combination ofthese methods. Means for preparing such polypeptides are well understoodin the art.

[0013] In a further aspect, the present invention relates to EPRG3ptpolynucleotides. Such polynucleotides include isolated polynucleotidescomprising a nucleotide sequence encoding a polypeptide which has atleast 70% identity, preferably at least 80% identity, more preferably atleast 90% identity, yet more preferably at least 95% identity, to theamino acid sequence of SEQ ID NO:2, over the entire length of SEQ IDNO:2. In this regard, polypeptides which have at least 97% identity arehighly preferred, whilst those with at least 98-99% identity are morehighly preferred, and those with at least 99% identity are most highlypreferred. Such polynucleotides include a polynucleotide comprising thenucleotide sequence contained in SEQ ID NO:1 encoding the polypeptide ofSEQ ID NO:2.

[0014] Further polynucleotides of the present invention include isolatedpolynucleotides comprising a nucleotide sequence that has at least 70%identity, preferably at least 80% identity, more preferably at least 90%identity, yet more preferably at least 95% identity, to a nucleotidesequence encoding a polypeptide of SEQ ID NO:2, over the entire codingregion. In this regard, polynucleotides which have at least 97% identityare highly preferred, whilst those with at least 98-99% identity aremore highly preferred, and those with at least 99% identity are mosthighly preferred.

[0015] Further polynucleotides of the present invention include isolatedpolynucleotides comprising a nucleotide sequence which has at least 70%identity, preferably at least 80% identity, more preferably at least 90%identity, yet more preferably at least 95% identity, to SEQ ID NO:1 overthe entire length of SEQ ID NO: 1. In this regard, polynucleotides whichhave at least 97% identity are highly preferred, whilst those with atleast 98-99% identity are more highly preferred, and those with at least99% identity are most highly preferred. Such polynucleotides include apolynucleotide comprising the polynucleotide of SEQ ID NO:1 as well asthe polynucleotide of SEQ ID NO:1.

[0016] The invention also provides polynucleotides which arecomplementary to all the above described polynucleotides.

[0017] The nucleotide sequence of SEQ ID NO:1 shows homology with humanp27 (Rasmussen, U.B., et al., 1993, Cancer Research 53:4096-4101). Thenucleotide sequence of SEQ ID NO:1 is a cDNA sequence and comprises apolypeptide encoding sequence (nucleotide 595 to 945) encoding apolypeptide of 116 amino acids, the polypeptide of SEQ ID NO:2. Thenucleotide sequence encoding the polvpeptide of SEQ ID NO:2 may beidentical to the polypeptide encoding sequence contained in SEQ ID NO:1or it may be a sequence other than the one contained in SEQ ID NO:1,which, as a result of the redundancy (degeneracy) of the genetic code,also encodes the polypeptide of SEQ ID NO:2. The polypeptide of SEQ IDNO:2 is structurally related to other proteins having homology and/orstructural similarity with human p27 (Rasmussen, U.B., et al., 1993,Cancer Research 53:4096-4101).

[0018] Preferred polypeptides and polynucleotides of the presentinvention are expected to have, inter alia, similar biologicalfunctions/properties to their homologous polypeptides andpolynucleotides. Furthermore, preferred polypeptides and polynucleotidesof the present invention have at least one EPRG3pt activity.

[0019] The present invention also relates to partial or otherpolynucleotide and polypeptide sequences which were first identifiedprior to the determination of the corresponding full length sequences ofSEQ ID NO:1 and SEQ ID NO:2.

[0020] Accordingly, in a further aspect, the present invention providesfor an isolated polynucleotide comprising:

[0021] (a) a nucleotide sequence which has at least 70% identity,preferably at least 80% identity, more preferably at least 90% identity,yet more preferably at least 95% identity, even more preferably at least97-99% identity to SEQ ID NO:3 over the entire length of SEQ ID NO:3;

[0022] (b) a nucleotide sequence which has at least 70% identity,preferably at least 80% identity, more preferably at least 90% identity,yet more preferably at least 95% identity, even more preferably at least97-99% identity, to SEQ ID NO:3 over the entire length of SEQ ID NO:3;or

[0023] (c) the polynucleotide of SEQ ID NO:3.

[0024] Accordingly, in a further aspect, the present invention providesfor an isolated polynucleotide comprising:

[0025] (a) a nucleotide sequence which has at least 70% identity,preferably at least 80% identity, more preferably at least 90% identity,yet more preferably at least 95% identity, even more preferably at least97-99% identity to SEQ ID NO:4 over the entire length of SEQ ID NO:4;

[0026] (b) a nucleotide sequence which has at least 70% identity,preferably at least 80% identity, more preferably at least 90% identity,yet more preferably at least 95% identity, even more preferably at least97-99% identity, to SEQ ID NO:4 over the entire length of SEQ ID NO:4;or

[0027] (c) the polynucleotide of SEQ ID NO:4.

[0028] The nucleotide sequences of SEQ ID NOS:3 and 4 and the peptidesequences encoded thereby are derived from EST (Expressed Sequence Tag)sequences. It is recognised by those skilled in the art that there willinevitably be some nucleotide sequence reading errors in EST sequences(see Adams, M. D. et al, Nature 377 (supp) 3, 1995). Accordingly, thenucleotide sequences of SEQ ID NOS:3 and 4 and the peptide sequencesencoded therefrom are therefore subject to the same inherent limitationsin sequence accuracy. Furthermore, the peptide sequences encoded by SEQID NOS:3 and 4 comprise a region of identity or close homology and/orclose structural similarity (for example a conservative amino aciddifference) with the closest homologous or structurally similar protein.

[0029] Polynucleotides of the present invention may be obtained, usingstandard cloning and screening techniques, from a cDNA library derivedfrom mRNA in cells of human bone marrow and hematopoietic cells, usingthe expressed sequence tag (EST) analysis (Adams, M. D., et al. Science(1991) 252:1651-1656; Adams, M. D. et al., Nature, (1992) 355:632-634;Adams, M. D., et al., Nature (1995) 377 Supp:3-174). Polynucleotides ofthe invention can also be obtained from natural sources such as genomicDNA libraries or can be synthesized using well known and commerciallyavailable techniques.

[0030] When polynucleotides of the present invention are used for therecombinant production of polypeptides of the present invention, thepolynucleotide may include the coding sequence for the maturepolypeptide, by itself; or the coding sequence for the maturepolypeptide in reading frame with other coding sequences, such as thoseencoding a leader or secretory sequence, a pre-, or pro- or prepro-protein sequence, or other fusion peptide portions. For example, amarker sequence which facilitates purification of the fused polypeptidecan be encoded. In certain preferred embodiments of this aspect of theinvention, the marker sequence is a hexa-histidine peptide, as providedin the pQE vector (Qiagen, Inc.) and described in Gentz et al., ProcNatl Acad Sci USA (1989) 86:821-824, or is an HA tag. The polynucleotidemay also contain non-coding 5′ and 3′ sequences, such as transcribed,non-translated sequences, splicing and polyadenylation signals, ribosomebinding sites and sequences that stabilize mRNA.

[0031] Further embodiments of the present invention includepolynucleotides encoding polypeptide variants which comprise the aminoacid sequence of SEQ ID NO:2 and in which several, for intance from 5 to10, 1 to 5, 1 to 3, 1 to 2 or 1, amino acid residues are substituted,deleted or added, in any combination.

[0032] Polynucleotides which are identical or sufficiently identical toa nucleotide sequence contained in SEQ ID NO:1, may be used ashybridization probes for cDNA and genomic DNA or as primers for anucleic acid amplification (PCR) reaction, to isolate full-length cDNAsand genomic clones encoding polypeptides of the present invention and toisolate cDNA and genomic clones of other genes (including genes encodinghomologs and orthologs from species other than human) that have a highsequence similarity to SEQ ID NO:1. Typically these nucleotide sequencesare 70% identical, preferably 80% identical, more preferably 90%identical, most preferably 95% identical to that of the referent. Theprobes or primers still generally comprise at least 15 nucleotides,preferably, at least 30 nucleotides and may have at least 50nucleotides. Particularly preferred probes will have betveen 30 and 50nucleotides.

[0033] A polynucleotide encoding a polypeptide of the present invention,including homologs and orthologs from species other than human, may beobtained by a process which comprises the steps of screening anappropriate library under stringent hybridization conditions with alabeled probe having the sequence of SEQ ID NO:1 or a fragment thereof;and isolating full-length cDNA and genomic clones containing saidpolynucleotide sequence. Such hybridization techniques are well known tothe skilled artisan. Preferred stringent hybridization conditionsinclude overnight incubation at 42° C. in a solution comprising: 50%formamide, 5xSSC (150 mM NaCl, 15 mNM trisodium citrate), 50 mM sodiumphosphate (pH7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20microgram/ml denatured, sheared salmon sperm DNA; followed by washingthe filters in 0.1x SSC at about 65° C. Thus the present invention alsoincludes polynucleotides obtainable by screening an appropriate libraryunder stingent hybridization conditions with a labeled probe having thesequence of SEQ ID NO:1 or a fragment thereof.

[0034] The skilled artisan will appreciate that, in many cases, anisolated cDNA sequence will be incomplete, in that the region coding forthe polypeptide is cut short at the 5′ end of the cDNA. This is aconsequence of reverse transcriptase, an enzyme with inherently low′processivity′ (a measure of the ability of the enzyme to remainattached to the template during the polymerisation reaction), failing tocomplete a DNA copy of the mRNA template during 1st strand cDNAsynthesis.

[0035] There are several methods available and well known to thoseskilled in the art to obtain full-length cDNAs, or extend short cDNAs,for example those based on the method of Rapid Amplification of cDNAends (RACE) (see, for example, Frohman et al., PNAS USA 85, 8998-9002,1988). Recent modifications of the technique, exemplified by theMarathon^(™) technology (Clontech Laboratories Inc.) for example, havesignificantly simplified the search for longer cDNAs. In theMarathon^(™) technology, cDNAs have been prepared from mRNA extractedfrom a chosen tissue and an ′adaptor′ sequence ligated onto each end.Nucleic acid amplification (PCR) is then carried out to amplify the′missing′ 5′ end of the cDNA using a combination of gene specific andadaptor specific oligonucleotide primers. The PCR reaction is thenrepeated using ′nested′ primers, that is, primers designed to annealwithin the amplified product (typically an adaptor specific primer thatanneals further 3′ in the adaptor sequence and a gene specific primerthat anneals further 5′ in the known gene sequence). The products ofthis reaction can then be analysed by DNA sequencing and a full-lengthcDNA constructed either by joining the product directly to the existingcDNA to give a complete sequence, or carrying out a separate full-lengthPCR using the new sequence information for the design of the 5′ primer.

[0036] Recombinant polypeptides of the present invention may be preparedby processes well known in the art from genetically engineered hostcells comprising expression systems. Accordingly, in a further aspect,the present invention relates to expression systems which comprise apolynucleotide or polynucleotides of the present invention, to hostcells which are genetically engineered with such expression sytems andto the production of polypeptides of the invention by recombinanttechniques. Cell-free translation systems can also be employed toproduce such proteins using RNAs derived from the DNA constructs of thepresent invention.

[0037] For recombinant production, host cells can be geneticallyengineered to incorporate expression systems or portions thereof forpolynucleotides of the present invention. Introduction ofpolynucleotides into host cells can be effected by methods described inmany standard laboratory manuals, such as Davis et al., Basic Methods inMolecular Biology (1986) and Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989). Preferred such methods include, forinstance, calcium phosphate transfection, DEAE-dexan mediatedtransfection, transvection, microinjection, cationic lipid-mediatedtransfection, electroporation, transduction, scrape loading, ballisticintroduction or infection.

[0038] Representative examples of appropriate hosts include bacterialcells, such as streptococci, staphylococci, E. coli, Streptomyces andBacillus subtilis cells; fungal cells, such as yeast cells andAspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 andBowes melanoma cells; and plant cells.

[0039] A great variety of expression systems can be used, for instance,chromosomal, episomal and virus-derived systems, e.g., vectors derivedfrom bacterial plasmids, from bacteriophage, from transposons, fromyeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses and retroviruses, and vectors derived from combinations thereof,such as those derived from plasmid and bacteriophage genetic elements,such as cosmids and phagemids. The expression systems may containcontrol regions that regulate as well as engender expression. Generally,any system or vector which is able to maintain, propagate or express apolynucleotide to produce a polypeptide in a host may be used. Theappropriate nucleotide sequence may be inserted into an expressionsystem by any of a variety of well-known and routine techniques, suchas, for example, those set forth in Sambrook et al., MOLECULAR CLONING,A LABORATORY MANUAL (supra). Appropriate secretion signals may beincorporated into the desired polypeptide to allow secretion of thetranslated protein into the lumen of the endoplasmic reticulum, theperiplasmic space or the extracellular environment. These signals may beendogenous to the polypeptide or they may be heterologous signals.

[0040] If a polypeptide of the present invention is to be expressed foruse in screening assays, it is generally preferred that the polypeptidebe produced at the surface of the cell. In this event, the cells may beharvested prior to use in the screening assay. If the polypeptide issecreted into the medium, the medium can be recovered in order torecover and purify the polypeptide. If produced intracellularly, thecells must first be lysed before the polypeptide is recovered.

[0041] Polypeptides of the present invention can be recovered andpurified from recombinant cell cultures by well-known methods includingammonium sulfate or ethanol precipitation, acid extraction, anion orcation exchange chromatography, phosphocellulose chromatography,hydrophobic interaction chromatography, affinity chromatography,hydroxylapatite chromatography and lectin chromatography. Mostpreferably, high performance liquid chromatography is employed forpurification. Well known techniques for refolding proteins may beemployed to regenerate active conformation when the polypeptide isdenatured during isolation and or purification.

[0042] This invention also relates to the use of polynucleotides of thepresent invention as diagnostic reagents. Detection of a mutated form ofthe gene characterised by the polynucleotide of SEQ ID NO:1 which isassociated with a dysfunction will provide a diagnostic tool that canadd to, or define, a diagnosis of a disease, or susceptibility to adisease, which results from under-expression, over-expression or alteredexpression of the gene. Individuals carrying mutations in the gene maybe detected at the DNA level by a variety of techniques.

[0043] Nucleic acids for diagnosis may be obtained from a subject'scells, such as from blood, urine, saliva, tissue biopsy or autopsymaterial. The genomic DNA may be used directly for detection or may beamplified enzymatically by using PCR or other amplification techniquesprior to analysis. RNA or cDNA may also be used in similar fashion.Deletions and insertions can be detected by a change in size of theamplified product in comparison to the normal genotype. Point mutationscan be identified by hybridizing amplified DNA to labeled EPRG3ptnucleotide sequences. Perfectly matched sequences can be distinguishedfrom mismatched duplexes by RNase digestion or by differences in meltingtemperatures. DNA sequence differences may also be detected byalterations in electrophoretic mobility of DNA fragments in gels, withor without denaturing agents, or by direct DNA sequencing (ee, e.g.,Myers et al., Science (1985)230:1242). Sequence changes at specificlocations may also be revealed by nuclease protection assays, such asRNase and S1 protection or the chemical cleavage method (see Cotton etal., Proc Nat Acad Sci USA (1985) 85: 4397-4401). In another embodimentan array of oligonucleotides probes comprising EPRG3pt nucleotidesequence or fragments thereof can be constructed to conduct efficientscreening of e.g., genetic mutations. Array technology methods are wellknown and have general applicability and can be used to address avariety of questions in molecular genetics including gene expression,genetic linkage, and genetic variability (see for example: M.Chee etal., Science, Vol 274, pp 610-613 (1996)).

[0044] The diagnostic assays offer a process for diagnosing ordetermining a susceptibility to the Diseases through detection ofmutation in the EPRG3pt gene by the methods described. In addition, suchdiseases may be diagnosed by methods comprising determining from asample derived from a subject an abnormally decreased or increased levelof polypeptide or mRNA. Decreased or increased expression can bemeasured at the RNA level using any of the methods well known in the artfor the quantitation of polynucleotides, such as, for example, nucleicacid amplification, for instance PCR, RT-PCR, RNase protection, Northernblotting and other hybridization methods. Assay techniques that can beused to determine levels of a protein, such as a polypeptide of thepresent invention, in a sample derived from a host are well-known tothose of skill in the art. Such assay methods include radioimmunoassays,competitive-binding assays, Western Blot analysis and ELISA assays.

[0045] Thus in arother aspect, the present invention relates to adiagonostic kit which comprises:

[0046] (a) a polynucleotide of the present invention, preferably thenucleotide sequence of SEQ ID NO:1, or a fragment thereof

[0047] (b) a nucleotide sequence complementary to that of (a);

[0048] (c) a polypeptide of the present invention, preferably thepolypeptide of SEQ ID NO:2 or a fragment thereof, or

[0049] (d) an antibody to a polypeptide of the present invention,preferably to the polypeptide of SEQ ID NO:2.

[0050] It will be appreciated that in any such kit, (a), (b), (c) or (d)may comprise a substantial component. Such a kit will be of use indiagnosing a disease or susceptibility to a disease, particularlyanemia, polycythemia, cancer, AIDS, and drug-induced anemias, amongstothers.

[0051] The nucleotide sequences of the present invention are alsovaluable for chromosome identification. The sequence is specificallytargeted to, and can hybridize with, a particular location on anindividual human chromosome. The mapping of relevant sequences tochromosomes according to the present invention is an important firststep in correlating those sequences with gene associated disease. Once asequence has been mapped to a precise chromosomal location, the physicalposition of the sequence on the chromosome can be correlated withgenetic map data. Such data are found in, for example, V. McKusick,Mendelian Inheritance in Man (available on-line through Johns HopkinsUniversity Welch Medical Library). The relationship between genes anddiseases that have been mapped to the same chromosomal region are thenidentified through linkage analysis (coinheritance of physicallyadjacent genes).

[0052] The differences in the cDNA or genomic sequence between affectedand unaffected individuals can also be determined. If a mutation isobserved in some or all of the affected individuals but not in anynormal individuals, then the mutation is likely to be the causativeagent of the disease.

[0053] The gene of the present invention maps to human chromosome14q31-q32. Lesions at this locus have been identified in some lymphoidmalignancies.

[0054] The polypeptides of the invention or their fragments or analogsthereof, or cells expressing them, can also be used as immunogens toproduce antibodies immunospecific for polypeptides of the presentinvention. The term “immunospecific” means that the antibodies havesubstantially greater affinity for the polypeptides of the inventionthan their affinity for other related polypeptides in the prior art.

[0055] Antibodies generated against polypeptides of the presentinvention may be obtained by administering the polypeptides orepitope-bearing fragments, analogs or cells to an animal, preferably anon-human animal, using routine protocols. For preparation of monoclonalantibodies, any technique which provides antibodies produced bycontinuous cell line cultures can be used. Examples include thehybridoma technique (Kohler. G. and Milstein, C., Nature (1975)256:495-497), the trioma technique, the human B-cell hybridoma technique(Kozbor et al., Immunology Today (1983) 4:72) and the EBV-hybridomatechnique (Cole et al., MONOCLONAL ANTIBODIES AND CANCER THERAPY, pp.77-96, Alan R. Liss, Inc., 1985).

[0056] Techniques for the production of single chain antibodies, such asthose described in U.S. Pat. No. 4,946,778, can also be adapted toproduce single chain antibodies to polypeptides of this invention. Also,transgenic mice, or other organisms, including other marmals, may beused to express humanized antibodies.

[0057] The above-described antibodies may be employed to isolate or toidentify clones expressing the polypeptide or to purify the polypeptidesby affinity chromatography.

[0058] Antibodies against polypeptides of the present invention may alsobe employed to treat the Diseases, amongst others.

[0059] In a further aspect, the present invention relates to geneticallyengineered soluble fusion proteins comprising a polypeptide of thepresent invention, or a fragment thereof, and various portions of theconstant regions of heavy or light chains of immunoglobulins of varioussubclasses (IgG, IgM, IgA, IgE). Preferred as an imrnmunoglobulin is theconstant part of the heavy chain of human IgG, particularly IgG1, wherefusion takes place at the hinge region. In a particular embodiment, theFc part can be removed simply by incorporation of a cleavage sequencewhich can be cleaved with blood clotting factor Xa. Furthermore, thisinvention relates to processes for the preparation of these fusionproteins by genetic engineering, and to the use thereof for drugscreening, diagnosis and therapy. A further aspect of the invention alsorelates to polynucleotides encoding such fusion proteins. Examples offusion protein technology can be found in International PatentApplication Nos. WO94/29458 and WO94/22914.

[0060] Another aspect of the invention relates to a method for inducingan immunological response in a mammal which comprises inoculating themammal with a polypeptide of the present invention, adequate to produceantibody and/or T cell immune response to protect said animal from theDiseases hereinbefore mentioned, amongst others. Yet another aspect ofthe invention relates to a method of inducing immunological response ina mammal which comprises, delivering a polypeptide of the presentinvention via a vector directing expression of the polynucleotide andcoding for the polypeptide in vivo in order to induce such animmunological response to produce antibody to protect said animal fromdiseases.

[0061] A further aspect of the invention relates to animmunological/vaccine formulation (composition) which, when introducedinto a mammalian host, induces an immunological response in that mammalto a polypeptide of the present invention wherein the compositioncomprises a polypeptide or polynucleotide of the present invention. Thevaccine formulation may further comprise a suitable carrier. Since apolypeptide may be broken down in the stomach, it is preferablyadministered parenterally (for instance, subcutaneous, intramuscular,intravenous, or intradermal injection). Formulations suitable forparenteral administration include aqueous and non-aqueous sterileinjection solutions which may contain anti-oxidants, buffers,bacteriostats and solutes which render the formulation instonic with theblood of the recipient; and aqueous and non-aqueous sterile suspensionswhich may include suspending agents or thickening agents. Theformulations may be presented in unit-dose or multi-dose containers, forexample, sealed ampoules and vials and may be stored in a freeze-driedcondition requiring only the addition of the sterile liquid carrierimmediately prior to use. The vaccine formulation may also includeadjuvant systems for enhancing the immunogenicity of the formulation,such as oil-in water systems and other systems known in the art. Thedosage will depend on the specific activity of the vaccine and can bereadily determined by routine experimentation.

[0062] Polypeptides of the present invention are responsible for manybiological functions, including many disease states, in particular theDiseases hereinbefore mentioned. It is therefore desirous to devisescreening methods to identify compounds which stimulate or which inhibitthe function of the polypeptide. Accordingly, in a further aspect, thepresent invention provides for a method of screening compounds toidentify those which stimulate or which inhibit the function of thepolypeptide. In general, agonists or antagonists may be employed fortherapeutic and prophylactic purposes for such Diseases as hereinbeforementioned. Compounds may be identified from a variety of sources, forexample, cells, cell-free preparations, chemical libraries, and naturalproduct mixtures. Such agonists, antagonists or inhibitors so-identifiedmay be natural or modified substrates, ligands, receptors, enzymes,etc., as the case may be, of the polypeptide; or may be structural orfunctional mimetics thereof (see Coligan et al., Current Protocols inImmunology 1(2):Chapter 5 (1991)).

[0063] The screening method may simply measure the binding of acandidate compound to the polypeptide, or to cells or membranes bearingthe polypeptide, or a fusion protein thereof by means of a labeldirectly or indirectly associated with the candidate compound.Alternatively, the screening method may involve competition with alabeled competitor. Further, these screening methods may test whetherthe candidate compound results in a signal generated by activation orinhibition of the polypeptide, using detection systems appropriate tothe cells bearing the polypeptide. Inhibitors of activation aregenerally assayed in the presence of a known agonist and the effect onactivation by the agonist by the presence of the candidate compound isobserved. Constitutively active polpypeptides may be employed inscreening methods for inverse agonists or inhibitors, in the absence ofan agonist or inhibitor, by testing whether the candidate compoundresults in inhibition of activation of the polypeptide. Further, thescreening methods may simply comprise the steps of mixing a candidatecompound with a solution containing a polypeptide of the presentinvention, to form a mixture, measuring EPRG3pt activity in the mixture,and comparing the EPRG3pt activity of the mixture to a standard. Fusionproteins, such as those made from Fc portion and EPRG3pt polypeptide, ashereinbefore described, can also be used for high-throughput screeningassays to identify antagonists for the polypeptide of the presentinvention (see D. Bennett et al., J Mol Recognition, 8:52-58 (1995); andK. Johanson et al., J Biol Chem, 270(16):9459-9471 (1995)).

[0064] The polynucleotides, polypeptides and antibodies to thepolypeptide of the present invention may also be used to configurescreening methods for detecting the effect of added compounds on theproduction of mRNA and polypeptide in cells. For example, an ELISA assaymay be constructed for measuring secreted or cell associated levels ofpolypeptide using monoclonal and polyclonal antibodies by standardmethods known in the art. This can be used to discover agents which mayinhibit or enhance the production of polypeptide (also called antagonistor agonist, respectively) from suitably manipulated cells or tissues.

[0065] The polypeptide may be used to identify membrane bound or solublereceptors, if any, through standard receptor binding techniques known inthe art. These include, but are not limited to, ligand binding andcrosslinking assays in which the polypeptide is labeled with aradioactive isotope (for instance, ¹²⁵I), chemically modified (forinstance, biotinylated), or fused to a peptide sequence suitable fordetection or purification, and incubated with a source of the putativereceptor (cells, cell membranes, cell supernatants, tissue extracts,bodily fluids). Other methods include biophysical techniques such assurface plasmon resonance and spectroscopy. These screening methods mayalso be used to identify agonists and antagonists of the polypeptidewhich compete with the binding of the polypeptide to its receptors, ifany. Standard methods for conducting such assays are well understood inthe art.

[0066] Examples of potential polypeptide antagonists include antibodiesor, in some cases, oligonucleotides or proteins which are closelyrelated to the ligands, substrates, receptors, enzymes, etc., as thecase may be, of the polypeptide, e.g., a fragment of the ligands,substrates, receptors, enzymes, etc.; or small molecules which bind tothe polypetide of the present invention but do not elicit a response, sothat the activity of the polypeptide is prevented.

[0067] Thus, in another aspect, the present invention relates to ascreening kit for identifying agonists, antagonists, ligands, receptors,substrates, enzymes, etc. for polypeptides of the present invention; orcompounds which decrease or enhance the production of such polypeptides,which comprises:

[0068] (a) a polypeptide of the present invention;

[0069] (b) a recombinant cell expressing a polypeptide of the presentinvention;

[0070] (c) a cell membrane expressing a polypeptide of the presentinvention; or

[0071] (d) antibody to a polypeptide of the present invention;

[0072] which polypeptide is preferably that of SEQ ID NO:2.

[0073] It will be appreciated that in any such kit, (a), (b), (c) or (d)may comprise a substantial component.

[0074] It will be readily appreciated by the skilled artisan that apolypeptide of the present invention may also be used in a method forthe structure-based design of an agonist, antagonist or inhibitor of thepolypeptide, by:

[0075] (a) determining in the first instance the three-dimensionalstructure of the polypeptide;

[0076] (b) deducing the three-dimensional structure for the likelyreactive or binding site(s) of an agonist, antagonist or inhibitor;

[0077] (c) synthesing candidate compounds that are predicted to bind toor react with the deduced binding or reactive site; and

[0078] (d) testing whether the candidate compounds are indeed agonists,antagonists or inhibitors.

[0079] It will be further appreciated that this will normally be aninterative process.

[0080] In a further aspect, the present invention provides methods oftreating abnormal conditions such as, for instance, anemia,polycythemia, cancer, AIDS, and drug-induced anemias, related to eitheran excess of, or an under-expression of, EPRG3pt polypeptide activity.

[0081] If the activity of the polypeptide is in excess, severalapproaches are available. One approach comprises administering to asubject in need thereof an inhibitor compound (antagonist) ashereinabove described, optionally in combination with a pharmaceuticallyacceptable carrier, in an arount effective to inhibit the function ofthe polypeptide, such as, for example, by blocking the binding ofligands, substrates, receptors, enzymes, etc., or by inhibiting a secondsignal, and thereby alleviating the abnormal condition. In anotherapproach, soluble forms of the polypeptides still capable of binding theligand, substrate, enzymes, receptors, etc. in competition withendogenous polypeptide may be administered. Typical examples of suchcompetitors include fragments of the EPRG3pt polypeptide.

[0082] In still another approach, expression of the gene encodingendogenous EPRG3pt polypeptide can be inhibited using expressionblocking techniques. Known such techniques involve the use of antisensesequences, either internally generated or separately administered (see,for example, O'Connor, J Neurochem (1991) 56:560 inOligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRCPress, Boca Raton, Fl. (1988)). Alternatively, oligonucleotides whichform triple helices with the gene can be supplied (see, for example, Leeet al., Nucleic Acids Res (1979) 6:3073; Cooney et al., Science(1988)241:456; Dervan et al., Science (1991)251:1360). These oligomerscan be administered per se or the relevant oligomers can be expressed invivo.

[0083] For treating abnormal conditions related to an under-expressionof EPRG3pt and its activity, several approaches are also available. Oneapproach comprises administering to a subject a therapeuticallyeffective amount of a compound which activates a polypeptide of thepresent invention, i.e., an agonist as described above, in combinationwith a pharmaceutically acceptable carrier, to thereby alleviate theabnormal condition. Alternatively, gene therapy may be employed toeffect the endogenous production of EPRG3pt by the relevant cells in thesubject. For example, a polynucleotide of the invention may beengineered for expression in a replication defective retroviral vector,as discussed above. The retroviral expression construct may then beisolated and introduced into a packaging cell transduced with aretroviral plasmid vector containing PNA encoding a polypeptide of thepresent invention such that the packaging cell now produces infectiousviral particles containing the gene of interest. These producer cellsmay be administered to a subject for engineering cells in vivo andexpression of the polypeptide in vivo. For an overview of gene therapy,see Chapter 20, Gene Therapy and other Molecular Genetic-basedTherapeutic Approaches, (and references cited therein) in HumanMolecular Genetics, T Strachan and A P Read, BIOS Scientific PublishersLtd (1996). Another approach is to administer a therapeutic amount of apolypeptide of the present invention in combination with a suitablepharmaceutical carrier.

[0084] In a further aspect, the present invention provides forpharmaceutical compositions comprising a therapeutically effectiveamount of a polypeptide, such as the soluble form of a polypeptide ofthe present invention, agonist/antagonist peptide or small moleculecompound, in combination with a pharmaceutically acceptable carrier orexcipient. Such carriers include, but are not limited to, saline,buffered saline, dextrose, water, glycerol, ethanol, and combinationsthereof. The invention further relates to pharmaceutical packs and kitscomprising one or more containers filled with one or more of theingredients of the aforementioned compositions of the invention.Polypeptides and other compounds of the present invention may beemployed alone or in conjunction with other compounds, such astherapeutic compounds.

[0085] The composition will be adapted to the route of administration,for intance by a systemic or an oral route. Preferred forms of systemicadministration include injection, typically by intravenous injection.Other injection routes, such as subcutaneous, intramuscular, orintraperitoneal, can be used. Alternative means for systemicadministration include transmucosal and transdermal administration usingpenetrants such as bile salts or fusidic acids or other detergents. Inaddition, if a polypeptide or other compounds of the present inventioncan be formulated in an enteric or an encapsulated formulation, oraladministration may also be possible. Administration of these compoundsmay also be topical and/or localized, in the form of salves, pastes,gels, and the like.

[0086] The dosage range required depends on the choice of peptide orother compounds of the present invention, the route of administration,the nature of the formulation, the nature of the subject's condition,and the judgment of the attending practitioner. Suitable dosages,however, are in the range of 0.1-100 μg/kg of subject. Wide variationsin the needed dosage, however, are to be expected in view of the varietyof compounds available and the differing efficiencies of various routesof administration. For example, oral administration would be expected torequire higher dosages than adinistration by intravenous injection.Variations in these dosage levels can be adjusted using standardempirical routines for optimization, as is well understood in the art.

[0087] Polypeptides used in treatment can also be generated endogenouslyin the subject, in treatment modalities often referred to as “genetherapy” as described above. Thus, for example, cells from a subject maybe engineered with a polynucleotide, such as a DNA or RNA, to encode apolypeptide ex vivo, and for example, by the use of a retroviral plasmidvector. The cells are then introduced into the subject.

[0088] Polynucleotide and polypeptide sequences form a valuableinformation resource with which to identify further sequences of similarhomology. This is most easily facilitated by storing the sequence in acomputer readable medium and then using the stored data to search asequence database using well known searching tools, such as GCC.Accordingly, in a further aspect, the present invention provides for acomputer readable medium having stored thereon a polynucleotidecomprising the sequence of SEQ ID NO:1 and/or a polypeptide sequenceencoded thereby.

[0089] The following definitions are provided to facilitateunderstanding of certain terms used frequently hereinbefore.

[0090] “Antibodies” as used herein includes polyclonal and monoclonalantibodies, chimeric, single chain, and humanized antibodies, as well asFab fragments, including the products of an Fab or other imnmunoglobulinexpression library.

[0091] “Isolated” means altered “by the hand of man” from the naturalstate. If an “isolated” composition or substance occurs in nature, ithas been changed or removed from its original environment, or both. Forexample, a polynucleotide or a polypeptide naturally present in a livinganimal is not “isolated,” but the same polynucleotide or polypeptideseparated from the coexisting materials of its natural state is“isolated”, as the term is employed herein.

[0092] “Polynucleotide” generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. “Polynucleotides” include, without limitation, single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions. Inaddition, “polynucleotide” refers to triple-stranded regions comprisingRNA or DNA or both RNA and DNA. The term “polynucleotide” also includesDNAs or RNAs containing one or more modified bases and DNAs or RNAs withbackbones modified for stability or for other reasons. “Modified” basesinclude, for example, tritylated bases and unusual bases such asinosine. A variety of modifications may be made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically or metabolicallymodified forms of polynucleotides as typically found in nature, as wellas the chemical forms of DNA and RNA characteristic of viruses andcells. “Polynucleotide” also embraces relatively short polynucleotides,often referred to as oligonucleotides.

[0093] “Polypeptide” refers to any peptide or protein comprising two ormore amino acids joined to each other by peptide bonds or modifiedpeptide bonds, i.e., peptide isosteres. “Polypeptide” refers to bothshort chains, commonly referred to as peptides, oligopeptides oroligomers, and to longer chains, generally referred to as proteins.Polypeptides may contain amino acids other than the 20 gene-encodedamino acids. “Polypeptides” include amino acid sequences modified eitherby natural processes, such as post-translational processing, or bychemical modification techniques which are well known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.Modifications may occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.It will be appreciated that the same type of modification may be presentto the same or varying degrees at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Polypeptides may be branched as a result of ubiquitination, and they maybe cyclic, with or without branching. Cyclic, branched and branchedcyclic polypeptides may result from post-translation natural processesor may be made by synthetic methods. Modifications include acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cystine, formation ofpyroglutamate, formylation, gamma-carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination (see, for instance, PROTEINS-STRUCTURE AND MOLECULARPROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, NewYork, 1993; Wold, F., Post-translational Protein Modifications:Perspectives and Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENTMODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, N.Y.,1983; Seifter et al., “Analysis for protein modifications and nonproteincofactors”, Meth Enzymol (1990) 182:626-646 and Rattan et al., “ProteinSynthesis: Post-translational Modifications and Aging”, Ann NY Acad Sci(1992) 663:48-62).

[0094] “Variant” refers to a polynucleotide or polypeptide that differsfrom a reference polynucleotide or polypeptide, but retains essentialproperties. A typical variant of a polynucleotide differs in nucleotidesequence from another, reference polynucleotide. Changes in thenucleotide sequence of the variant may or may not alter the amino acidsequence of a polypeptide encoded by the reference polynucleotide.Nucleotide changes may result in amino acid substitutions, additions,deletions, fusions and truncations in the polypeptide encoded by thereference sequence, as discussed below. A typical variant of apolypeptide differs in amino acid sequence from another, referencepolypeptide. Generally, differences are limited so that the sequences ofthe reference polypeptide and the variant are closely similar overalland, in many regions, identical. A variant and reference polypeptide maydiffer in amino acid sequence by one or more substitutions, additions,deletions in any combination. A substituted or inserted amino acidresidue may or may not be one encoded by the genetic code. A variant ofa polynucleotide or polypeptide may be a naturally occurring such as anallelic variant, or it may be a variant that is not known to occurnaturally. Non-naturally occurring variants of polynucleotides andpolypeptides may be made by mutagenesis techniques or by directsynthesis.

[0095] “Identity,” as known in the art, is a relationship between two ormore polypeptide sequences or two or more polynucleotide sequences, asthe case may be, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”can be readily calculated by known methods, including but not limited tothose described in (Computational Molecular Biology, Lesk, A. M., ed.,Oxford University Press, N.Y., 1988; Biocomputing: Informatics andGenome Projects, Smith, D. W., ed., Academic Press, N.Y., 1993; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, N.J., 1994; Sequence Analysis in Molecular Biology,von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer,Gribskov, M. and Devereux, J., eds., M Stockton Press, N.Y., 1991; andCarillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988).Methods to determine identity are designed to give the largest matchbetween the sequences tested. Moreover, methods to determine identityare codified in publicly available computer programs. Computer programmethods to determine identity between two sequences include, but are notlimited to, the GCG program package (Devereux, J., et al., Nucleic AcidsResearch 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S.F. etal., J. Molec. Biol. 215: 403-410 (1990). The BLAST X program ispublicly available from NCBI and other sources (BLAST Manual, Altschul,S., et al, NCBI NLM NIH Bethesda, MD 20894; Altschul, S., et al., J.Mol. Biol. 215: 403-410 (1990). The well known Smith Waterman algorithmmay also be used to determine identity.

[0096] Parameters for polypeptide sequence comparison include thefollowing:

[0097] 1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453(1970)

[0098] Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc.Natl. Acad. Sci. USA. 89:10915-10919 (1992)

[0099] Gap Penalty: 12

[0100] Gap Length Penalty: 4

[0101] A program useful with these parameters is publicly available asthe “gap” program from Genetics Computer Group, Madison Wis. Theaforementioned parameters are the default parameters for peptidecomparisons (along with no penalty for end gaps).

[0102] Parameters for polynucleotide comparison include the following:

[0103] 1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453(1970)

[0104] Comparison matrix: matches=+10, mismatch=0

[0105] Gap Penalty: 50

[0106] Gap Length Penalty: 3

[0107] Available as: The “gap” program from Genetics Computer Group,Madison Wis. These are the default parameters for nucleic acidcomparisons.

[0108] A preferred meaning for “identity” for polynucleotides andpolypeptides, as the case may be, are provided in (1) and (2) below.

[0109] (1) Polynucleotide embodiments further include an isolatedpolynucleotide comprising a polynucleotide sequence having at least a50, 60, 70, 80, 85, 90, 95, 97 or 100% identity to the referencesequence of SEQ ID NO:1, wherein said polynucleotide sequence may beidentical to the reference sequence of SEQ ID NO:1 or may include up toa certain integer number of nucleotide alterations as compared to thereference sequence, wherein said alterations are selected from the groupconsisting of at least one nucleotide deletion, substitution, includingtransition and transversion, or insertion, and wherein said alterationsmay occur at the 5′ or 3′ terminal positions of the reference nucleotidesequence or anywhere between those terminal positions, interspersedeither individually among the nucleotides in the reference sequence orin one or more contiguous groups within the reference sequence, andwherein said number of nucleotide alterations is determined bymultiplying the total number of nucleotides in SEQ ID NO:1 by theinteger defining the percent identity divided by 100 and thensubtracting that product from said total number of nucleotides in SEQ IDNO:1, or:

n_(n)≦x_(n)−(x_(n)•y),

[0110] wherein n_(n) is the number of nucleotide alterations, x_(n) isthe total number of nucleotides in SEQ ID NO:1, y is 0.50 for 50%, 0.60for 60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95for 95%, 0.97 for 97% or 1.00 for 100%, and • is the symbol for themultiplication operator, and wherein any non-integer product of x_(n)and y is rounded down to the nearest integer prior to subtracting itfrom x_(n). Alterations of a polvnucleotide sequence encoding thepolypeptide of SEQ ID NO:2 may create nonsense, missense or frameshiftmutations in this coding sequence and thereby alter the polypeptideencoded by the polynucleotide following such alterations.

[0111] By way of example, a polynucleotide sequence of the presentinvention may be identical to the reference sequence of SEQ ID NO:2,that is it may be 100% identical, or it may include up to a certaininteger number of amino acid alterations as compared to the referencesequence such that the percent identity is less than 100% identity. Suchalterations are selected from the group consisting of at least onenucleic acid deletion, substitution, including transition andtransversion, or insertion, and wherein said alterations may occur atthe 5′ or 3′ terminal positions of the reference polynucleotide sequenceor anywhere between those terminal positions, interspersed eitherindividually among the nucleic acids in the reference sequence or in oneor more contiguous groups within the reference sequence. The number ofnucleic acid alterations for a given percent identity is determined bymultiplying the total number of amino acids in SEQ ID NO:2 by theinteger defining the percent identity divided by 100 and thensubtracting that product from said total number of amino acids in SEQ IDNO:2, or:

n_(n)≦x_(n)−(x_(n)•y),

[0112] wherein n_(n) is the number of amino acid alterations, x_(n) isthe total number of amino acids in SEQ ID NO:2, y is, for instance 0.70for 70%, 0.80 for 80%, 0.85 for 85% etc., • is the symbol for themultiplication operator, and wherein any non-integer product of x_(n)and y is rounded down to the nearest integer prior to subtracting itfrom x_(n).

[0113] (2) Polypeptide embodiments further include an isolatedpolypeptide comprising a polypeptide having at least a 50,60, 70, 80,85, 90, 95, 97 or 100% identity to a polypeptide reference sequence ofSEQ ID NO:2, wherein said polypeptide sequence may be identical to thereference sequence of SEQ ID NO: 2 or may include up to a certaininteger number of amino acid alterations as compared to the referencesequence, wherein said alterations are selected from the groupconsisting of at least one amino acid deletion, substitution, includingconservative and non-conservative substitution, or insertion and whereinsaid alterations may occur at the amino- or carboxy-terminal positionsof the reference polypeptide sequence or anywhere between those terminalpositions, interspersed either individually among the amino acids in thereference sequence or in one or more contiguous groups within thereference sequence, and wherein said number of amino acid alterations isdetermuned by multiplying the total number of amino acids in SEQ ID NO:2by the integer defining the percent identity divided by 100 and thensubtracting that product from said total number of amino acids in SEQ IDNO:2, or:

n_(a)≦x_(a)−(x_(a)•y),

[0114] wherein n_(a) is the number of amino acid alterations, x_(a) isthe total number of amino acids in SEQ ID NO:2, y is 0.50 for 50%, 0.60for 60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95for 95%, 0.97 for 97% or 1.00 for 100%, and • is the symbol for themultiplication operator, and wherein any non-integer product of x_(a)and y is rounded down to the nearest integer prior to subtracting itfrom x_(a).

[0115] By way of example, a polypeptide sequence of the presentinvention may be identical to the reference sequence of SEQ ID NO:2,that is it may be 100% identical, or it may include up to a certaininteger number of amino acid alterations as compared to the referencesequence such that the percent identity is less than 100% identity. Suchalterations are selected from the group consisting of at least one aminoacid deletion, substitution, including conservative and non-conservativesubstitution, or insertion, and wherein said alterations may occur atthe amino- or carboxy-terminal positions of the reference polypeptidesequence or anywhere between those terminal positions, interspersedeither individually among the amino acids in the reference sequence orin one or more contiguous groups within the reference sequence. Thenumber of amino acid alterations for a given % identity is determined bymultiplying the total number of amino acids in SEQ ID NO:2 by theinteger defining the percent identity divided by 100 and thensubtracting that product from said total number of amino acids in SEQ IDNO:2, or:

n_(a)≦x_(a)−(x_(a)•y),

[0116] wherein n_(a) is the number of amino acid alterations, x_(a) isthe total number of amino acids in SEQ ID NO:2, y is, for instance 0.70for 70%, 0.80 for 80%, 0.85 for 85% etc., and • is the symbol for themultiplication operator, and wherein any non-integer product of x_(a)and y is rounded down to the nearest integer prior to subtracting itfrom x_(a).

[0117] “Fusion protein” refers to a protein encoded by two, oftenunrelated, fused genes or fragments thereof. In one example, EP-A-0 464discloses fusion proteins comprising various portions of constant regionof immunoglobulin molecules together with another human protein or partthereof. In many cases, employing an immunoglobulin Fc region as a partof a fusion protein is advantageous for use in therapy and diagnosisresulting in, for example, improved pharmacokinetic properties [see,e.g., EP-A 0232 262]. On the other hand, for some uses it would bedesirable to be able to delete the Fc part after the fusion protein hasbeen expressed, detected and purified.

[0118] All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as if each individual publication were specifically andindividually indicated to be incorporated by reference herein as thoughfully set forth.

[0119] On northern blots of UT7-EPO cells, EPRG3pt is observed as a 3 kbEPO-inducible message. EPRG3pt is induced by EPO in the presence of theprotein synthesis inhibitor, cycloheximide, even though there is noinduction of EPRG3pt when cycloheximide alone is added. Theseobservations show EPRG3pt to be an EPO primary response gene, whoseinduction of expression is not dependent on new protein synthesis.Induction of EPRG3pt expression must therefore occur through directactivation of signalling pathway(s) by EPO. The treatment withEPO+cycloheximide apparently leads to superinduction of EPRG3pt messagelevels, because stimulation with EPO alone did not result in detectablelevels of EPRG3pt message. Induction of erythroid differentiation ofUT7-EPO cells by hemin treatment leads to lower expression of the 3 kbspecies and increased amounts of smaller messages.

[0120] Examination of PCR amplified cDNA from human bone marrow cellsshows induction of expression of EPRG3pt as a 1.1 kb species when thecells are cultured with EPO+stem cell factor (SCF), and to lesserextent, after culture with IL3+SCF+flt3 ligand. EPRG3pt was undetectablein the untreated cells, and after culture with TPO+SCF and G−CSF+SCF.Northern blots containing RNA from human bone marrow cells treated withfactors also exhibited strong induction of a 1.2 kb message for EPRG3ptwhen the cell's treatment included EPO.

[0121] Limited expression of EPRG3pt has been found by probing northernblots with RNA from other tissues. In addition, some different messagesizes were found. The highest levels occur in spleen (4.8, 4.2 kb),placenta (4.8, 0.9 kb), and peripheral blood leukocytes (0.9 kb). SEQ IDNO:1GTGGCATTCAAGCATTCTATAGTGCTTGCCGTGACTCAGGCGCACAGTGCAAAAGGAAQCACATCTTTCTCTGCCATGAGGACTTtATTAGTGTCTGAAGAGCtTTTTCTGGACTATAGGAGAAAGTCATGGTCTCCCTCACTAATAAACACTGACCCTGCTTCGGATGAGCTAACAGCCCTGCTCAGAAAGCATGACACCCATCCTGTTCACTTTTCCCTGCCCCGGTCAAACCACCAAGCCCTAGATTTCATTGCCAAGTATCCTGTATTTACTGGGAATGGACATCACAGTAGCATGCCTTCCTAGCCACATCTATGAGGTTTTGTTCATTTTCATTCTGCTTTTTGTTTGAGCTAAACCTGCCTTGGGAGGCAGAAAAGAAAAGATAACATCTGATTCCCCCGATCAACCAACCAATCAACTAGGTCAGGGCCACGTAAATTCATTCAGGACaAGCACTGAGGTCAAACTCCCCAGTGATCCTCACCCTCCTCCAGAATTTCCACTTCCCGAAATGAAGCAAAGAGCGGTAGACAGGAGTCATCCCTTCTTGTGGCTCCCAACcTGGGGCAGCCCCCTGCCTCCCTTTAGATGGGCAATCGGCTTAGaAAGtGGAGGGGAAGCCAGTGTGGATCTACTCACAGAATGTTCTTTTGGTTTCCAGCCAGGATTGCTACAGTTGTGATTGGAGGAGTTGTggccatggcGGCTGTOCCCATGGTGCTCAGTGCCATGGGCTTCACTGCGGCGGGAATCGCCTCGTCCTCCATAGCAGCCAAGATGATGTCCGCGGCGGCCATTGCCAATGGGGGTGGAGTTGCCTCGGGCAGCCTTGTGGCTACTCTGCAGTCACTGGGAGCAACTGGACTCTCCGGATTGACCAAGTTCATCCTGGGCTCCATTGGGTCTGCCATTGCGGCTGTCATTGCGAGGTTCTACTAGCTCCCTGCCCCTCGCCCTOCAGAGAAGAGAACCATGCCAGGGGAGAAGGCACCCAGCCATCCTGACCCAGcGAGGAGCCAACTATCCCAAATATACCTGGGGTGAAATATACCAAATTTTGCATTTCCAGAQGAAAATAAGAAATAAAGATGAATTGTTGCAACAn

[0122] SEQ ID NO:2MGNRLRKWRGSQCGSTHRMFFWFPARIATVVIGGVVAMAAVPMVLSAMGFTAAGIASSSIAAKMMSAAAIANGGGVASGSLVATLQSLGATGLSGLTKFILGSIGSAIAA VIARFY*

[0123] SEQ ID NO:3   1 AGCACTCTCC AGCCTCTCAC CGCAATCCAC ACTGGCTTCCCCTCCGCTTC  51 CTAAGCCGAT TGCCCATCTA AAGGGAGGCA GGGGGCTGCC CCAAgTTGGG101 AGCCACAAGA AGGGATGACT CCTGTCTACC GCTCTTTGCT TCATTTCGGG 151AAGTGGAAAT TCTGGAGGAG GGTGAGGATC

[0124] SEQ ID NO:4GATCCACACTGGCTTCCCCTCCGCTTCCTAAGCCGATTGCCCATCTAAAGGGAGGCAGGGGGCTGCCCCAAgTTGGGAGCCACAAGAAGGGATGACTCCTGTCTACCGCTCTTTGCTTCATTTCGGGAAGTGGAAATTCTGGAGGAGGGTGAGGATC

[0125]

1 4 1 1111 DNA HOMO SAPIENS UNSURE (1111) 1 gtggcattca agcattctatagtgcttgcc gtgactcagg cgcacagtgc aaaaggaagc 60 acatctttct ctgccatgaggactttatta gtgtctgaag agctttttct ggactatagg 120 agaaagtcat ggtctccctcactaataaac actgaccctg cttcggatga gctaacagcc 180 ctgctcagaa agcatgacacccatcctgtt cacttttccc tgccccggtc aaaccaccaa 240 gccctagatt tcattgccaagtatcctgta tttactggga atggacatca cagtagcatg 300 ccttcctagc cacatctatgaggttttgtt cattttcatt ctgctttttg tttgagctaa 360 acctgccttg ggaggcagaaaagaaaagat aacatctgat tcccccgatc aaccaaccaa 420 tcaactaggt cagggccacgtaaattcatt caggacaagc actgaggtca aactccccag 480 tgatcctcac cctcctccagaatttccact tcccgaaatg aagcaaagag cggtagacag 540 gagtcatccc ttcttgtggctcccaacctg gggcagcccc ctgcctccct ttagatgggc 600 aatcggctta gaaagtggaggggaagccag tgtggatcta ctcacagaat gttcttttgg 660 tttccagcca ggattgctacagttgtgatt ggaggagttg tggccatggc ggctgtgccc 720 atggtgctca gtgccatgggcttcactgcg gcgggaatcg cctcgtcctc catagcagcc 780 aagatgatgt ccgcggcggccattgccaat gggggtggag ttgcctcggg cagccttgtg 840 gctactctgc agtcactgggagcaactgga ctctccggat tgaccaagtt catcctgggc 900 tccattgggt ctgccattgcggctgtcatt gcgaggttct actagctccc tgcccctcgc 960 cctgcagaga agagaaccatgccaggggag aaggcaccca gccatcctga cccagcgagg 1020 agccaactat cccaaatatacctggggtga aatataccaa attttgcatt tccagaggaa 1080 aataagaaat aaagatgaattgttgcaaca n 1111 2 116 PRT HOMO SAPIENS 2 Met Gly Asn Arg Leu Arg LysTrp Arg Gly Ser Gln Cys Gly Ser Thr 1 5 10 15 His Arg Met Phe Phe TrpPhe Pro Ala Arg Ile Ala Thr Val Val Ile 20 25 30 Gly Gly Val Val Ala MetAla Ala Val Pro Met Val Leu Ser Ala Met 35 40 45 Gly Phe Thr Ala Ala GlyIle Ala Ser Ser Ser Ile Ala Ala Lys Met 50 55 60 Met Ser Ala Ala Ala IleAla Asn Gly Gly Gly Val Ala Ser Gly Ser 65 70 75 80 Leu Val Ala Thr LeuGln Ser Leu Gly Ala Thr Gly Leu Ser Gly Leu 85 90 95 Thr Lys Phe Ile LeuGly Ser Ile Gly Ser Ala Ile Ala Ala Val Ile 100 105 110 Ala Arg Phe Tyr115 3 180 DNA HOMO SAPIENS 3 agcactctcc agcctctcac cgcaatccac actggcttcccctccgcttc ctaagccgat 60 tgcccatcta aagggaggca gggggctgcc ccaagttgggagccacaaga agggatgact 120 cctgtctacc gctctttgct tcatttcggg aagtggaaattctggaggag ggtgaggatc 180 4 157 DNA HOMO SAPIENS 4 gatccacact ggcttcccctccgcttccta agccgattgc ccatctaaag ggaggcaggg 60 ggctgcccca agttgggagccacaagaagg gatgactcct gtctaccgct ctttgcttca 120 tttcgggaag tggaaattctggaggagggt gaggatc 157

What is claimed is:
 1. An isolated polypeptide selected from the groupconsisting of: (i) an isolated polypeptide comprising an amino acidsequence selected from the group having at least: (a) 70% identity; (b)80% identity; (c) 90% identity; or (d) 95% identity to the amino acidsequence of SEQ ID NO:2 over the entire length of SEQ ID NO:2; (ii) anisolated polypeptide comprising the amino acid sequence of SEQ ID NO:2or (iii) an isolated polypeptide which is the amino acid sequence of SEQID NO:2.
 2. An isolated polynucleotide selected from the groupconsisting of (i) an isolated polynucleotide comprising a nucleotidesequence encoding a polypeptide that has at least (a) 70% identity; (b)80% identity; (c) 90% identity; or (d) 95% identity; to the amino acidsequence of SEQ ID NO:2, over the entire length of SEQ ID NO:2; (ii) anisolated polynucleotide comprising a nucleotide sequence that has atleast: (a) 70% identity (b) 80% identity; (c) 90% identity; or (d) 95%identity; over its entire length to a nucleotide sequence encoding thepolypeptide of SEQ ID NO:2; (iii) an isolated polynucleotide comprisinga nucleotide sequence which has at least: (a) 70% identity; (b) 80%identity; (c) 90% identity; or (d) 95%identity; to that of SEQ ID NO:1over the entire length of SEQ ID NO: 1; (iv) an isolated polynucleotidecomprising a nucleotide sequence encoding the polypeptide of SEQ IDNO:2; (vi) an isolated polynucleotide which is the polynucleotide of SEQID NO:1; or (vi) an isolated polynucleotide obtainable by screening anappropriate library under stringent hybridization conditions with alabeled probe having the sequence of SEQ ID NO:1 or a fragment thereof;or a nucleotide sequence complementary to said isolated polynucleotide.3. An antibody immunospecific for the polypeptide of claim
 1. 4. Amethod for the treatment of a subject: (i) in need of enhanced activityor expression of the polypeptide of claim 1 comprising: (a)administering to the subject a therapeutically effective amount of anagonist to said polypeptide; and/or (b) providing to the subject anisolated polynucleotide comprising a nucleotide sequence encoding saidpolypeptide in a form so as to effect production of said polypeptideactivity in vivo.; or (ii) having need to inhibit activity or expressionof the polypeptide of claim 1 comprising: (a) administering to thesubject a therapeutically effective amount of an antagonist to saidpolypeptide; and/or (b) administering to the subject a nucleic acidmolecule that inhibits the expression of a nucleotide sequence encodingsaid polypeptide; and/or (c) administering to the subject atherapeutically effective amount of a polypeptide that competes withsaid polypeptide for its ligand, substrate, or receptor.
 5. A processfor diagnosing a disease or a susceptibility to a disease in a subjectrelated to expression or activity of the polypeptide of claim 1 in asubject comprising: (a) determining the presence or absence of amutation in the nucleotide sequence encoding said polypeptide in thegenome of said subject; and/or (b) analyzing for the presence or amountof said polvpeptide expression in a sample derived from said subject. 6.A method for screening to identify compounds which stimulate or whichinhibit the function of the polypeptide of claim 1 which comprises amethod selected from the group consisting of: (a) measuring the bindingof a candidate compound to the polypeptide (or to the cells or membranesbearing the polypeptide) or a fusion protein thereof by means of a labeldirectly or indirectly associated with the candidate compound; (b)measuring the binding of a candidate compound to the polypeptide (or tothe cells or membranes bearing the polypeptide) or a fusion proteinthereof in the presence of a labeled competitor; (c) testing whether thecandidate compound results in a signal generated by activation orinhibition of the polypeptide, using detection systems appropriate tothe cells or cell membranes bearing the polypeptide; (d) mixing acandidate compound with a solution containing a polypeptide of claim 1,to form a mixture, measuring activity of the polypeptide in the mixture,and comparing the activity of the mixture to a standard; or (e)detecting the effect of a candidate compound on the production of mRNAencoding said polypeptide and said polypeptide in cells, using forinstance, an ELISA assay.
 7. An agonist or an antagonist of thepolypeptide of claim
 1. 8. An expression system comprising apolynucleotide capable of producing a polypeptide of claim 1 when saidexpression system is present in a compatible host cell.
 9. A process forproducing a recombinant host cell comprising transforming ortransfecting a cell with the expression system of claim 8 such that thehost cell, under appropriate culture conditions, produces a polypeptidecomprising an amino acid sequence having at least 70% identity to theamino acid sequence of SEQ ID NO:2 over the entire length of SEQ IDNO:2.
 10. A recombinant host cell produced by the process of claim 9.11. A membrane of a recombinant host cell of claim 10 expressing apolypeptide comprising an amino acid sequence having at least 70%identity to the amino acid sequence of SEQ ID NO:2 over the entirelength of SEQ ID NO:2.
 12. A process for producing a polypeptidecomprising culturing a host cell of claim 10 under conditions sufficientfor the production of said polypeptide and recovering the polypeptidefrom the culture.
 13. An isolated polynucleotide selected form the groupconsisting of: (a) an isolated polynucleotide comprising a nucleotidesequence which has at least 70%, 80%, 90%, 95%, 97% identity to SEQ IDNO:3 over the entire length of SEQ ID NO:3; (b) an isolatedpolynucleotide comprising the polynucleotide of SEQ ID NO:3; or (c) thepolynucleotide of SEQ ID NO:3.
 14. An isolated polynucleotide selectedform the group consisting of: (a) an isolated polynucleotide comprisinga nucleotide sequence which has at least 70%, 80%, 90%, 95%, 97%identity to SEQ ID NO:4 over the entire length of SEQ ID NO:4; (b) anisolated polynucleotide comprising the polynucleotide of SEQ ID NO:4; or(c) the polynucleotide of SEQ ID NO:4.